Cutaneous inflammation accelerates the premalignant expansion of melanocytes bearing oncogenic mutations

Reviewer #1 (Public review):

Summary:

The immune system represents a source for melanocyte-extrinsic determinants of melanoma. Multiple immune cell types, including natural killer cells and CD8+ cytotoxic T lymphocytes, destroy cancer cells directly, and this anti-tumor activity is widely thought to eliminate many nascent tumors before they become clinically detectable. Regulatory T (Treg) cells, which are defined by expression of the transcription factor Foxp3, function as critical suppressors of lymphocyte activation in both homeostatic and disease contexts. Intratumoral Treg cell accumulation has been associated with disease progression in the clinic, and Treg cell depletion inhibits melanoma outgrowth in transplantable models of the disease.

However, the role of Treg cells during the early, premalignant stage of melanocyte expansion has not been examined. To study the interplay between incipient melanoma and cutaneous inflammation, the authors subjected an autochthonous murine model of melanoma [LSL-BrafV600E;Ptenfl/fl;Tyr::CreERT2 (BPT)mice] to three distinct inflammatory immune perturbations. Each of these perturbations accelerated premalignant melanocyte outgrowth, which was unexpected given that both Treg cell depletion and DNFB treatment markedly enhanced conventional T (Tconv) cell infiltration into the skin. Detailed analysis of each inflammatory response revealed a shared cellular and molecular signature comprising myeloid infiltration, characteristic cytokines and tissue remodeling factors, and vascular permeability. Altogether, the results support the hypothesis that oncogenic mutations in melanocytes along with altered immune response and inflammation synergistically drive melanomagenesis.

Strengths:

(1) The use of the three distinct inflammatory immune perturbations, such as transient Treg cell depletion, acute UV- B irradiation, and 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity.

(2) The re-examination of the role of Treg cells in transplantable models of tumor growth by Subcutaneous (s.c.) implantation of syngeneic B16F10 melanoma cells as widely used to study anti-tumor immunity and to interrogate the effects of Treg cells on tumor suppression.

(3) The use of the LSL-TdTomato mice for measuring the TdTomato fluorescence in each immune cell type to assess uptake of melanocyte antigen.

(4) scRNA-seq data document a broad and rapid myeloid inflammatory response in Treg cell-deficient skin.

Weaknesses:

(1) The expression of inflammatory mediators Il1b, Il6, and TNFa, and angiogenic mediators Hif1a and Ang2 in all of the three models of immune perturbation has been verified at the transcript level by qRT-PCR, and it remains to be determined whether it correlates with the same at the protein level.

(2) scRNA-seq data to profile the diversity of immune cells (CD45+) in the ear skin of the DNFB-treated contact hypersensitivity model are currently missing.

(3) The authors indicate that at least 2 prior studies directly implicated inflammatory macrophages in the melanocyte proliferation response. However, no attempts were made for the identification of the UVB-driven factors underlying myeloid recruitment by which these cells activate melanocytes.

(4) It is very surprising to observe that altered immune responses, such as enhanced Tconv cell priming and activation in Treg cell-deficient skin, failed to antagonize mutant melanocyte outgrowth in the BPT model. While UVB-induced skin inflammation differs somewhat from the response to Treg cell depletion, both feature the infiltration of tissue remodeling macrophages and vascular instability. The findings that contact hypersensitivity can promote the expansion of non-malignant BRAF (V600E)Pten- Het melanocytes have interesting implications for benign hyperpigmentation conditions, such as post-inflammatory hyperpigmentation, Riehl's melanosis, and melasma.

Reviewer #2 (Public review):

Summary:

Tran and colleagues investigate how inflammation alters the earliest stages of melanoma tumorigenesis in mice carrying LSL-BrafV600E, Ptenfl/fl, and Tyr-CreERT2 alleles. They compare transient regulatory T cell depletion, acute UVB irradiation, and DNFB-induced contact hypersensitivity. Each perturbation increases ear pigmentation and Tyrp1 expression after oncogene induction. The inflammatory settings also share recruitment of monocytes and macrophages, expression of inflammatory and tissue-remodeling programs, and increased vascular permeability. Dexamethasone attenuates the DNFB-associated phenotype. A secondary finding of particular interest is that regulatory T cell depletion accelerates the premalignant BPT phenotype but inhibits B16F10 tumor growth, suggesting that regulatory T cells can have different effects during tumor initiation and established transplantable disease.

The study addresses an important question that is difficult to approach using transplantable tumor models. The data convincingly show that each perturbation produces substantial inflammation in the skin and that vascular leakage accompanies the response. At present, though, the central biological endpoint is not sufficiently separated from melanogenesis. Darkening of the ear and increased Tyrp1 RNA can reflect more pigment or altered differentiation within the existing oncogene-carrying melanocytes rather than an increase in their number, particularly given that pigment content is itself variable in transformed melanocytes, which range from heavily pigmented to nearly amelanotic. This issue is especially important in the UVB and DNFB experiments, where inflammatory signals can alter pigmentation directly.

Strengths:

The autochthonous BPT model is a major strength. It preserves the native relationship between melanocytes and the surrounding stromal and immune compartments during lesion initiation. Including three distinct inflammatory perturbations makes the recurring association with melanocyte-associated readouts more persuasive than any single model would be. The paired-ear DNFB design is efficient and controls for inter-animal variability. The combination of flow cytometry, single-cell RNA sequencing, intravital imaging, and Evans Blue assays provides useful complementary evidence that the inflammatory interventions remodel the local tissue environment. The B16F10 experiments help establish that the unexpected effect of regulatory T cell depletion is specific to the early autochthonous setting rather than a general failure of the depletion model. The BT-Het experiment is also thoughtful in asking whether inflammation can enhance the phenotype of oncogene-carrying melanocytes in a nevus-stage context that does not proceed to full malignant progression after oncogene induction alone.

Weaknesses:

The strongest caveat concerns the central claim. The outgrowth readouts are ear darkening and bulk Tyrp1 expression, but both may report pigment or differentiation state rather than the number of oncogene-carrying melanocytes. Pigment content is not a reliable proxy for cell number here, since the same population can darken or lighten without any change in cell number. No direct count or lineage-reporter measurement is provided for the regulatory T cell, UVB, or DNFB comparisons. Until that gap is filled, the data support increased pigmentation of oncogene-carrying melanocytes more firmly than the premalignant expansion named in the title, and this concern is most pronounced in the UVB and DNFB settings, where inflammation can change pigmentation on its own.

Secondly, the proposed shared mechanism is largely associative. Dexamethasone appropriately shows that inflammation as a whole is required for the DNFB phenotype, but as a broad anti-inflammatory it cannot isolate any single component. The manuscript singles out blood vessel remodeling as particularly important, and that specific attribution exceeds what a non-selective drug can show, especially as no individual pathway is selectively blocked in a tumor-initiation experiment and Il6 is reduced only modestly. The authors acknowledge that the precise chain of causation is unresolved, so the vascular claim should be softened to match or tested directly.

Also, several of the mechanistic conclusions rest on thin or single cohorts and on single-cell data whose replication is not fully reported, making them less convincing than the inflammatory phenotypes themselves. The systemic regulatory T cell model shows the consequences of body-wide depletion rather than a skin-specific regulatory T cell function, and the inferred monocyte-to-macrophage trajectory reflects transcriptional similarity rather than a demonstrated lineage path. The interpretation of dendritic-cell TdTomato uptake as evidence of antigen presentation or T cell priming is not supported by a direct measure of reactivity.

Finally, the nevus-stage framing should be corrected. The manuscript frames the BT-Het experiment as testing non-oncogenic conditions, but those melanocytes carry BrafV600E, so it is better read as inflammation-enhanced behavior of oncogene-carrying melanocytes at the nevus stage.

Reviewer #3 (Public review):

Summary:

Tran et. al. investigate how inflammation in the skin influences the early stages of melanomagenesis. They use an autochthonous, tamoxifen-inducible mouse melanoma model (LSL-BrafV600E;Ptenfl/fl;Tyr::CreERT2, "BPT") to examine three inflammatory perturbations: transient depletion of regulatory T cells, acute ultraviolet-B irradiation, and contact hypersensitivity induced by 2,4-dinitrofluorobenzene (DNFB). They report that each perturbation promotes the recruitment of immune cells, especially inflammatory monocytes and macrophages, increased expression of inflammatory and tissue-remodeling factors, and enhanced vascular permeability, which ultimately increases the outgrowth of premalignant melanocytes measured by local pigmentation and expression of the melanocyte-associated gene Tyrp1. In the DNFB model, the authors showed that treatment with dexamethasone reduces the effects of contact hypersensitivity on pigmentation, inflammatory gene expression, and vascular leakage, potentially providing a translational angle.

Strengths:

An interesting observation is that transient Treg depletion promotes premalignant melanocyte outgrowth in the autochthonous BPT model while inhibiting the growth of transplantable B16 F10 tumors. This contrast is consistent with a role for Tregs in limiting inflammatory disruption of the skin during early tumorigenesis and highlights the value of autochthonous models. These findings may also have broader implications for understanding the stage- and context-dependent functions of Tregs in cancer.

Another strength of this manuscript is the comparison of three mechanistically distinct inflammatory perturbations. Treg depletion, UVB irradiation, and DNFB-induced contact hypersensitivity engage different inflammatory pathways but converge on myeloid-cell recruitment, inflammatory gene expression, and increased vascular permeability. This convergence strengthens the conclusion that an acute inflammatory microenvironment is associated with enhanced melanocyte outgrowth during the early premalignant phase.

Weaknesses:

The paper convincingly establishes a correlation between the inflammatory signature and melanocyte outgrowth across three distinct perturbations. However, the mechanistic claim that myeloid cells and/or vascular remodeling drive melanocyte expansion rests primarily on the dexamethasone experiments in the DNFB model. Because dexamethasone broadly affects immune, stromal, endothelial, and melanocytic compartments, these experiments do not establish that inflammatory monocytes/macrophages or vascular destabilization are specifically required for the melanocyte response.

A related limitation is that the proposed monocytic origin of the inflammatory macrophage population following perturbation remains inferred. Although the scRNA-seq data and pseudotime analysis in Figure 4 - Supplement 2 are consistent with a trajectory from monocytes to macrophages, they do not exclude local reprogramming of resident macrophages into an inflammatory state. This alternative is particularly relevant because resident macrophage populations have been implicated in vascular remodeling and tumor outgrowth (PMIDs: 36493773 and 40216154).

Finally, the assessment of melanocyte outgrowth is largely through increased pigmentation and whole-ear Tyrp1 expression. Although these measurements may reflect increased melanocyte abundance, they may also be influenced by melanogenic activity or increased Tyrp1 expression per cell. More direct evidence of melanocyte proliferation, such as Ki67 or EdU/BrdU staining specifically within TdTomato-positive melanocytes, would support the use of "expansion" and "proliferation" throughout the manuscript. Histopathological characterization of lesion architecture, atypia, proliferation, and invasion would also help establish the premalignant nature of the lesions.